The National Science Foundation provided funding used in part for this for this invention under grant RF 5311. Accordingly, the Federal Government may have certain rights in this invention pursuant to 35 U.S.C .sctn. 202.
1. Field of the Invention
The invention relates to an expression vector system based on the regulation of bacterial luminescence (the positive feedback lux regulatory circuit). The invention further relates to the construction of a precisely regulatable expression vector system which comprises a complete luxR gene in combination with an inactivated luxI gene, both of which are under the control of a common control region. The invention allows the precise temporal expression of gene products otherwise deleterious or lethal to the cell when controlled by standard expression systems. The invention further relates to the control of the expression system of the invention by an inexpensive inducer.
2. Description of the Related Art
Numerous expression systems exist for expression of gene products in bacteria. However, cloning and expression of genes which have deleterious effects on or which kill the cells in which they are expressed represents a continuing problem. Among these problem genes are a wide array of genes whose effects on the cell range from mildly deleterious to those gene products which are lethal to the cell in even minute quantities. As used herein, a deleterious gene is any gene whose expression in host cells in culture would prevent that culture from achieving the normal logarithmic growth which the culture would achieve but for the expression of the deleterious gene. Furthermore, as used herein, an expression system which is capable of stringently controlling the expression of such deleterious genes is an expression system which can sufficiently limit the expression of the deleterious gene in host cells in culture in order to allow the culture to achieve normal logarithmic growth which the culture would not achieve without the stringent control due to some level of transcription of the deleterious gene. Thus, in the case of genes whose products are lethal to the cell in even minute quantity, stringent control is that level of control which almost completely eliminates transcription of the lethal gene until released from that control.
Some deleterious genes encode gene products which if expressed in limited quantity are actually useful to the cell while if expressed in even slightly elevated quantities are deleterious to the cell. For example, such genes are epitomized by DNA-modifying enzymes such as the DNA restriction enzymes used throughout molecular biology. If allowed to be expressed in a host which is not resistant to the restriction enzyme, the host cell's own DNA is susceptible to degradation by the cloned gene's product (Rosenberg et al. 1981).
Even where a particular gene product merely stresses the host cell by its presence or by its overabundance, the production of these proteins in the cell may not be feasible. Such an effect has been observed, for instance, with overproduction of several of the subunits of E. coli RNA polymerase. Although it has been possible to overproduce the RNA polymerase subunits in host cells, their overexpression causes a reduction in growth rate of cultures of these cells (Bedwell and Nomura, 1986). These mildly deleterious effects represent enough of a stress to the population of cells that cells within that population, which contain a mutated version of the desired protein whose production has fewer or no deleterious effects compared to the non-mutated protein, may overgrow those cells containing the wild type protein.
Even more deleterious gene products include many proteins which become associated with the membrane of the host cells, some of which effect the cell to such a degree that the host cells are killed (Michaelis and Beckwith 1982). Some genes, in fact, code for proteins which, if expressed to any degree, even at levels as low as a few molecules of the protein per cell, lead quickly to the death of the host cell. These type of genes are typified by the class of genes encoding the lysis proteins of viruses (e.g., .lambda. lysis protein or MS2 lysis protein) and the lytic proteins of bacteria (e.g., colicins of certain enteric bacteria) (Coleman et al. 1983; Adhya et al. 1971; Konisky 1982).
A number of patented expression systems for use in bacterial hosts have been described. In some cases, the expression systems relate to generalized expression systems. In others, specific positive regulation systems have been described. Other patented expression systems have been designed to allow relatively tight regulation. In some instances, these expression systems were individually tailored for expression of a particular protein which presented some difficulty using standard expression systems.
For instance, U.S. Pat. No. 4,782,022 appears to relate to the construction of a vector comprising a promoter of a constitutively expressed gene coupled to a gene which codes for a product capable of activating other genes required for nitrogen fixation. U.S. Pat. No. 4,775,630 appears to relate to a variant of an adenovirus control region, the regulator of which is especially sensitive to repression by products of the gene under its control. U.S. Pat. No. 4,767,708 appears to relate to the construction of a recombinant vector containing a cloned bacterial DNA polymerase I under operable control of a conditionally controllable foreign promoter. This patent notes that the foreign promoter may be a positively regulated promoter. The invention appears to be designed to overproduce DNA polymerase. U.S. Pat. No. 4,677,064 appears to relate to the use of the promoters of bacteriophage .lambda., P.sub.L and N.sub.RBS, in order to construct a vector capable of overexpressing human tumor necrosis factor. U.S. Pat. No. 4,634,678 appears to relate to construction of a variety of expression vectors all of which are based upon negative control systems. The patent's specification does, however, suggest the replacement of negative control sequences with positive control sequences. U.S. Pat. No. 4,578,355 appears to relate to the use of the P.sub.L promoter of bacteriophage .lambda. to construct a high level expression vector. U.S. Pat. No. 4,503,142 appears to relate to the construction of a class of cloning and expression vectors capable of heterologous gene expression. These vectors are based on the use of the lac promoter/operator of Escherichia coli (E. coli).
All of these systems suffer, to greater or lesser degrees, from the inability to control expression to the extent required when the gene product will kill or otherwise seriously damage the host cell if expressed. The analogy can be drawn to an electrical switch connected functionally to a device capable of inflicting great harm to those which encounter it, even if the amount of electricity reaching the device is minimal. The design electrical engineer would find it most unsatisfactory if the only switches available were those which constantly fed the lethal device small amounts of power. Moreover, even where the prior art expression systems have provided a means for limited expression of certain deleterious genes, the likelihood that the gene will mutate in order to prevent the deleterious effects on the host cell from being realized has always caused concern. This is especially true where large scale operations have been envisioned.
Additionally, many of the prior art expression systems must rely for induction of expression either on the host's biochemical responses or on costly or awkward induction means. Moreover, many prior art expression systems suffer from the fact that the inducer is a compound routinely found in nature such as naturally occurring sugar compounds. Thus, great care must be taken to prevent inadvertent exposure of cells to extraneous sources of such commonly encountered inducers.
The present inventors are involved in research into regulation of bioluminescence in the marine bacterium Vibrio fischeri, which regulation has been studied extensively through cloning and genetic manipulation of the lux system in E. coli (Devine et al. 1989; Dunlap and Greenberg 1985; Dunlap and Greenberg 1988; Engebrecht et al. 1983; Engebrecht and Silverman 1984; Engebrecht and Silverman 1986). Expression of the lux genes in V. fischeri is controlled by a unique form of positive feedback regulation called autoinduction, and this pattern of regulation may be duplicated by the cloned system in E. coli (Engebrecht et al. 1983; Engebrecht and Silverman 1986). The autoinduction response is mediated by the production and accumulation of a small molecule, the autoinducer, which is synthesized in the presence of the luxI gene product. This product molecule presumably interacts with the luxR gene product to induce the synthesis of the enzymes required for light production. Kaplan and Greenberg (1987) were able to overproduce the luxR gene product in E. coli, develop a procedure for purifying this overproduced protein, but were unable to demonstrate convincingly that LuxR protein had DNA-binding activity.
The autoinducer of V. fischeri has been identified as N-(3-oxo-hexanoyl) homoserine lactone (Eberhard et al. 1981) and has been shown to be both freely diffusible across the cytoplasmic membrane and species specific in its ability to stimulate bioluminescence (Eberhard 1972; Kaplan and Greenberg 1985). This molecule has been synthesized in vitro and shown to function in a biological assay (Eberhard et al. 1981; Kaplan et al. 1985).
The lux genes are organized into two divergently transcribed operons, termed rightward and leftward, which are separated by a common regulatory region (Devine et al. 1988; Engrebrecht et al. 1983; Engebrecht and Silverman 1987). The luxR gene is the only known gene in the leftward operon (operon.sub.L) and encodes a positive regulatory protein which, in the presence of autoinducer, stimulates transcription of the rightward operon (operon.sub.R). This interaction has recently been shown to require the 20-base-pair lux operator located in the control region (Devine et al. 1989). Operon.sub.R consists of at least six genes (luxICDABE). The luxI gene encodes a protein required for autoinducer synthesis (Engebrecht and Silverman 1984), the luxC, luxD, and luxE genes encode enzymes which provide luciferase with an aldehyde substrate (Meighen 1988), and the luxA and luxB genes encode the .alpha. and .beta. subunits of the luciferase enzyme. The sequence of the entire lux regulon from V. fisheri has been determined (Baldwin et al. 1989).
The current model describing the autoinduction process suggests that a low basal level of transcription of operon.sub.R leads to low-level synthesis of autoinducer by luxI. High cell density is required for autoinducer to accumulate, since it is freely diffusible across the cytoplasmic membrane. It is by virtue of the diffusible nature of autoinducer that the expression of luminescence is, in nature, cell density-dependent. If the LuxR protein, whose synthesis is regulated at the transcriptional level by the cyclic AMP-catabolite gene activator protein (cAMP-CAP) system (Dunlap and Greenberg 1985; Dunlap and Greenberg 1988), has also accumulated, it can form a complex with autoinducer capable of binding to the lux operator and stimulating transcription of operon.sub.R. Positive feedback results from the presence of luxI in operon.sub.R, since stimulation of rightward transcription of luxR and autoinducer leads to the production of more autoinducer by increased levels of LuxI protein. In addition to this primary regulatory circuit, several global regulatory systems in E. coli have been shown to interact with the lux system to affect the timing of induction of bioluminescence including the heat shock (.sigma..sup.32) system and the SOS response (Ulitzur 1989; Ulitzer and Kuhn 1988). Thus, the positive feedback mechanism of the lux regulatory circuitry leads to the sharp induction of the enzymes required for light production.
Expression systems are needed which do not rely for their induction on expensive or otherwise inadequate induction mechanisms. This is especially important for commercial operation of bacterial fermentations of useful gene products. More importantly, however, expression systems are needed which are capable of very stringently regulating the expression of deleterious or lethal genes until such time as induction of expression can be used to express commercial quantities of their otherwise harmful gene products. If such systems were available, the expression and genetic manipulation of a wide array of otherwise lethal or deleterious gene products would be possible via the powerful capabilities of batch fermentation.